Method for predicting and treating clinically significant prostate cancer

ABSTRACT

The present invention relates to methods, devices, combinations, kits, and systems for predicting and treating clinically significant prostate cancer in a urine sample of an individual suspected of suffering from prostate cancer based on expression analysis of normalised prostate tumour markers. The present methods, devices, combinations, kits, and systems are especially suitable for predicting and treating prostate cancer with a Gleason score of seven or more in individuals with a serum prostate-specific antigen (sPSA) level lower than 15 ng/ml. Specifically, the present invention relates to methods, devices, combinations, kits, and systems for predicting and treating clinically significant prostate cancer in a urine sample of an individual suspected of suffering from prostate cancer, the method comprises the steps of: a) determining mRNA expression levels of one or more of the genes DLX1, HOXC6, TDRD1 and KLK3 in a urine sample of said individual; b) normalizing the mRNA expression levels of one ore more of DLX1, HOXC6, and TDRD1 using the mRNA expression level of KLK3; and c) establishing clinically significant prostate cancer based on a combination of the KLK3 normalized expression levels of the combination of one or more of DLX1, HOXC6, and TDRD1. Treatment for prostate cancer may be administered based on the expression levels of one or more of DLX1, HOXC6, and TDRD1.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.119(e) to U.S. Provisional Patent Application No. 62/258,163, filed onNov. 20, 2016, the content of which is incorporated herein by referencein its entirety.

BACKGROUND

The present invention relates to methods for predicting clinicallysignificant prostate cancer in a biopsy using a urine sample of anindividual suspected of suffering from prostate cancer. It is based onexpression analysis of normalised prostate tumour markers. The presentmethods are especially suitable for predicting prostate cancer with aGleason score of seven or more in individuals with a serumprostate-specific antigen (sPSA) level lower than 15 ng/ml. The presentinvention further relates to kits of parts for predicting clinicallysignificant prostate cancer in a urine sample based on expressionanalysis and to the use of normalised prostate tumour markers fordetecting prostate cancer in a urine sample.

Worldwide, prostate cancer or PCa is the second most frequentlydiagnosed cancer among men, with 1.1 million estimated new cases and307.500 estimated deaths in 2012. Since the introduction of serumprostate-specific antigen (sPSA) testing, the incidence of PCa hasincreased. However, sPSA-testing has also led to an increased amount ofunnecessary biopsies and diagnosis of clinically insignificant tumorswhich would not have been life-threatening (potential overtreatment);especially in the sPSA ‘grey-zone’ (4.0-10.0 ng/ml) where 65-70% of menhave a negative biopsy result. Men with indolent disease, who undergotreatment, may suffer complications without a reduction in their risk ofdying from PCa. Generally, men with high-grade PCa have a highprobability of dying from PCa within 10 years, whereas men withlow-grade PCa have a minimal risk of dying from this disease.

The major challenge is to improve the detection of clinicallysignificant or high-grade PCa in an early stage. Both overdiagnosis andovertreatment could be reduced if PCa-specific biomarkers coulddistinguish indolent from aggressive tumors. Ideally the biomarkerscould be measured in a sample that can be obtained non-invasively, forexample in urine.

The Prostate CAncer gene 3 (PCA3)-based urinary test (Progensa® PCA3,GenProbe) is the only FDA-approved molecular diagnostic test for thedetection of PCa in urine. PCA3 was identified as a gene encoding a longnon-coding RNA that was consistently upregulated in PCa. PCA3 was shownto be of value in PCa detection, however, the relation with tumoraggressiveness and thus prognostic value remains controversial.

A stepwise approach for the identification and selection of newbiomarkers using gene expression profiling has been suggested. A genepanel measured in urinary sediments predicted Gleason score≧7 uponprostate biopsy. The test, based on mRNA levels of Homeobox C6 (HOXC6),Distal-less Homeobox 1 (DLX1), and Tudor domain containing 1 (TDRD1),was shown to have independent additional value to sPSA for predictinghigh-grade PCa upon biopsy. The combination of HOXC6, DLX1, and TDRD1outperformed Progensa® PCA3. Furthermore, the predictive accuracy couldbe improved when urinary HOXC6, DLX1, and TDRD1 were combined with sPSA.HOXC6, DLX1, and TDRD1 are upregulated in PCa and these genes may beinvolved in the onset of PCa and are associated with high-grade PCa.However, although the test showed promising results when performed on aurine sediment sample, analysis of whole urine samples were notsatisfactory.

SUMMARY

Considering the above, there is an urgent clinical unmet need in the artfor new biomarkers that can predict high-grade disease to aid indecision making regarding further diagnostic evaluations (for exampleprostate biopsies or imaging) and treatment.

It is an object of the present invention, amongst other objects, to meetthe above need in the art.

According to the invention this object is met, amongst other objected asoutlined in the appended claims.

Specifically, this object, amongst other objects, is, according to afirst aspect, met by method for predicting clinically significantprostate cancer in a urine sample of an individual suspected ofsuffering from prostate cancer, the methods comprise the steps of:

-   -   a1) determining mRNA expression levels of one or more of the        genes DLX1, HOXC6, TDRD1, and KLK3 in a urine sample of said        individual;    -   b1) normalizing the mRNA expression levels of one or more of        DLX1, HOXC6, and TDRD1 using the mRNA expression level of KLK3        (e.g., by dividing the expression levels of one or more of DLX1,        HOXC6, and TDRD1 by the mRNA expression level of KLK3);    -   c) establishing clinically significant prostate cancer based on        a combination of the KLK3 normalized expression levels of the        combination of DLX1 and HOXC6 and optionally TDRD1.

DLX1 belongs to the family of homeodomain transcription factors whichare related to the Drosophila distal-less (Dll) gene. The family hasbeen related to a number of developmental features and appears to bewell preserved across species. Dlx genes are implicated in tangentialmigration of interneurons from the subpallium to the pallium duringvertebrate brain development. It has been suggested that Dlx promotesthe migration of interneurons by repressing a set of proteins that arenormally expressed in terminally differentiated neurons and act topromote the outgrowth of dendrites and axons.

With respect to DLX1 expression, at least two transcript variants areknown. Transcript variant 1 is longer than transcript variant 2 andcontains an internal exon in the coding region that results in a frameshift and premature stop codon. Within the context of the presentinvention, DLX1 expression level determination refers to determinationof the expression levels of both transcripts.

HOXC6 is a family member of the homeobox superfamily of genes and theHOX subfamily contain members that are transcription factors involved incontrolling and coordinating complex functions during development viaspatial and temporal expression patterns. In humans, there are 39classical HOX genes organized into the clusters A, B, C and D.

Three genes, HOXC4, HOXC5 and HOXC6, share a 5′ non-coding exon.Transcripts may include the shared exon spliced to the gene-specificexons, or they may include only the gene-specific exons. For HOXC6,alternatively spliced transcript variants encoding different isoformshave been identified. Transcript variant two represents the longertranscript and includes the shared exon. It contains a distinct 5′UTRand lacks an in-frame portion of 5′ coding region compared to variantone. The resulting isoform two has a shorter N-terminus when compared toisoform one. Transcript variant one includes only gene-specific exonsand encodes the longer isoform. Within the context of the presentinvention, HOXC6 expression level determination refers to the expressionlevels of variants 1 and 2.

Prostate-specific antigen (PSA), also known as gamma-semino protein orkallikrein-3 (KLK3), is a glycoprotein enzyme encoded by the KLK3 gene.KLK3 is a member of the kallikrein-related peptidase family and issecreted by the epithelial cells of the prostate gland. KLK3 is producedfor the ejaculate, where it liquefies semen in the seminal coagulum andallows sperm to swim freely. KLK3 is present in small quantities in theserum of men with healthy prostates, but is often elevated in thepresence of prostate cancer or other prostate disorders.

It is noted that the United States Preventive Services Task Force(USPSTF, 2012) does not recommend PSA screening, noting that the testmay result in “overdiagnosis” and “overtreatment” because “most prostatecancer is asymptomatic for life” and treatments involve risks ofcomplications including impotence (erectile dysfunction) andincontinence. The USPSTF concludes “the potential benefit does notoutweigh the expected harms.” Approximately 30 percent of patients withhigh KLK3 have prostate cancer diagnosed after biopsy.

According to a preferred embodiment, the present mRNA expression levelsof DLX1 and HOXC6 and optionally TDRD1 are normalised by calculating theratio between DLX1 mRNA, HOXC6 mRNA and optionally TDRD1 mRNA and theexpression level of KLK3 mRNA.

According to another preferred embodiment, the present mRNA expressionlevels are determined using a quantitative Polymerase Chain Reaction(qPCR). The use of qPCR allows for, when determining mRNA expressionlevels, to measure the crosspoint cycle (Cp), i.e. the cycle wherein thedetection signal crosses a predetermined threshold. The Cp valueinversely correlates with the initial concentration of the mRNA, e.g.the lower the Cp the higher the gene expression in the sample. The Cpvalues and copy numbers of the present target genes allows forcalculating the present ratios to normalize the qPCR results relative toa reference level for example by using the Delta DeltaCp method (ΔΔCp):

Ratio = 2((AvgCp_((Cal 10⁴)_(target)) − Cp  _((sample)_(target))) − (AvgCp_((Cal 10⁴)) − Cp_((sample)_(reference)))) × 10.000

A suitable reference level for normalizing the expression levels of atarget (e.g., DLX1, HOXC2, and/or TDRD1 mRNA) may include the expressionlevel of KLK3 mRNA.

According to the present invention, a clinically significant prostatecancer preferably is high-grade prostate cancer or prostate cancer witha Gleason score of 7 or higher. The Gleason grading system is generallyused to evaluate the prognosis of men with prostate cancer using samplesfrom a prostate biopsy. Together with other parameters, it isincorporated into a strategy of prostate cancer staging which predictsprognosis and helps guide therapy. A Gleason score is given to prostatecancer based upon its microscopic appearance. Cancers with a higherGleason score are more aggressive and have a worse prognosis.

According to yet another preferred embodiment, the present method isused for predicting clinically significant prostate cancer in a urinesample of an individual having a serum prostate-specific antigen (sPSA)level lower than 15 ng/ml, preferably lower than 10 ng/ml. Especially inthis group of individuals, the chance of “overdiagnosis” and“overtreatment” is relatively high

According to still another preferred embodiment, the present methodfurther comprises

-   -   a) determining mRNA expression levels of the genes HOXC4 and/or        TDRD1 in a urine sample of said individual;    -   b) normalizing the mRNA expression levels of HOXC4 and/or TDRD1        using the mRNA expression level of KLK3; and        step c comprises establishing clinically significant prostate        cancer based on a combination of the KLK3 normalized expression        levels of the combination of DLX1 and HOXC6 and HOXC4 and/or        TDRD1.

The present determining mRNA expression levels preferably comprisesisolating mRNA from a urine sample preferably a first voided urinesample.

According to a second aspect, the present invention relates to kit ofparts for predicting clinically significant prostate cancer comprising:

-   -   1) one or more means for collecting urine;    -   2) means for isolating mRNA from urine; and    -   3) means for determining mRNA expression levels of the genes        DLX1, HOXC6 and KLK3.        And, according to a third aspect, to the use of mRNA expression        levels of HOXC6 and DLX1 and optionally TDRD1 in a urine sample        for predicting clinically significant prostate cancer wherein        said mRNA expression levels of HOXC6 and DLX1 and optionally        TDRD1 are normalised against KLK3 mRNA expression levels.

The foregoing methods further may include performing the foregoingmethods to determine the mRNA expression levels of the genes DLX1 andHOXC6 and optionally TDRD1 in a urine sample and/or requesting a testproviding results of an analysis to determine the mRNA expression levelsof the genes DLX1 and HOXC6 and optionally TDRD1 in a urine sample. Inaddition, the foregoing methods may include performing furtherdiagnostic tests and/or requesting results of further diagnostic tests,based on the detected mRNA expression levels of HOXC6 and/or DLX1 and/orTDRD1 (e.g., based on detecting elevated or reduced expression levels ofHOXC and/or DLX1 and/or TDRD1 relative to a normal control which mayinclude nucleic acid isolated from urine of a patient not havingprostate cancer). Further, the foregoing methods may includeadministering therapy for prostate cancer based on the detected mRNAexpression levels of HOXC6 and/or DLX1 and/or TDRD1 (e.g., based ondetecting elevated or reduced expression levels of HOXC and/or DLX1and/or TDRD1 relative to a normal control which may include nucleic acidisolated from urine of a patient not having prostate cancer).

The disclosed methods may be performed utilizing devices, combinations,kits, and systems that comprise or utilize components for detecting mRNAexpression levels of one or more of HOXC6, DLX1, TDRD1 and/or KLK3 in aurine sample. In addition, the disclosed methods may be performedutilizing devices, combinations, kits, and systems that comprise orutilize components for treating prostate cancer based the detected mRNAexpression levels of one or more of HOXC6, DLX1, TDRD1 and/or KLK3 in aurine sample.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further detailed in the following exampleof an especially preferred embodiment of the present invention. In theexample, reference is made to figures wherein:

FIG. 1: shows the ROC curves for the prediction of high grade PCa uponprostate biopsy: (1) DLX1/KLK3 ratio (black solid line; AUC 0.64 [95%CI: 0.58-0.71]); (2) DLX1 copy number (grey dotted line; AUC 0.51 [95%CI: 0.45-0.58]);

FIG. 2: shows the ROC curves for the prediction of high grade PCa uponprostate biopsy: (1) HOXC6/KLK3 ratio (black solid line; AUC 0.72 [95%CI: 0.67-0.78]); (2) HOXC6 copy number (grey dotted line; AUC 0.54 [95%CI: 0.48-0.61]);

FIG. 3: shows the ROC curves for the prediction of high grade PCa uponprostate biopsy: (1) HOXC6−DLX1 score (black solid line; AUC 0.75 [95%CI: 0.70-0.80]); (2) PCA3 (grey dotted line; AUC 0.65 [95% CI:0.59-0.71]);

FIG. 4: shows the HOXC6−DLX1 score in relation to the Gleason score incohort A (A) and cohort B (B). Mann-Whitney U test, significance levelp<0.05; and

FIG. 5: shows the predictive accuracy (AUC) of the HOXC6−DLX1 score andsPSA for the detection of high grade PCa at low sPSA subgroups forcohort A (A) and cohort B (B).

DETAILED DESCRIPTION

Disclosed are methods, devices, combinations, kits, and systems fordiagnosing and treating prostate cancer. The methods, devices,combinations, kits, and systems are described herein using severaldefinitions, as set forth below and throughout the application.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise. For example, “a component” should be interpreted to mean “oneor more components” unless the context clearly dictates otherwise.

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

The presently disclosed methods, devices, combinations, kits, andsystems relate to detecting elevated expression of mRNA of the genesDLX1 and/or HOXC6 in a urine sample in order to diagnose and/or prognosean individual, and optionally treat the diagnosed and/or prognosedindividual by administering therapy to the individual for treatingprostate cancer based on the genetic marker having been identified.Elevated expression of mRNA of the genes DLX1 and/or HOXC6 may beidentified relative to an internal control (e.g., expression of mRNA ofthe gene KLK3) and/or relative to an external control (e.g., expressionof mRNA of the genes DLX1 and/or HOXC6 in a patient not having prostatecancer). Expression of mRNA of the genes DLX1 and/or HOXC6 in a urinesample may be normalized relative to expression of mRNA of the gene KLK3in the urine sample and a HOXC6−DLX1 score may be calculated asdisclosed herein (e.g., a HOXC6−DLX1 score of at least about 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 or higher). Further diagnostics may be performed on a samplefrom the individual based on the HOXC6−DLX1 score (e.g., a Gleason scoreon a prostate biopsy from the patient) and/or therapy may beadministered to the patient based on the HOXC6−DLX1 score. A HOXC6−DLX1score may be generated by determining the area under the curve (AUC) ofa Receiver Operating Characteristic (ROC) curve and corresponding 95%confidence intervals (CI).

Optionally, the presently disclosed methods, devices, combinations,kits, and systems may relate to detecting elevated expression of mRNA ofthe genes DLX1, HOXC6, and/or TDRD1 in a urine sample in order todiagnose and/or prognose an individual, and optionally treat thediagnosed and/or prognosed individual by administering therapy to theindividual for treating prostate cancer based on the genetic markerhaving been identified. Elevated expression of mRNA of the genes DLX1,HOXC6, and/or TDRD1 may be identified relative to an internal control(e.g., expression of mRNA of the gene KLK3) and/or relative to anexternal control (e.g., expression of mRNA of the genes DLX1, HOXC6,and/or TDRD1 in a patient not having prostate cancer). Expression ofmRNA of the genes DLX1, HOXC6, and/or TDRD1 in a urine sample may benormalized relative to expression of mRNA of the gene KLK3 in the urinesample and a HOXC6-DLX1-TDRD1 score may be calculated as disclosedherein (e.g., a HOXC6−DLX1-TDRD1 score of at least about 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 or higher). Further diagnostics may be performed on a samplefrom the individual based on the HOXC6−DLX1-TDRD1 score (e.g., a Gleasonscore on a prostate biopsy from the patient) and/or therapy may beadministered to the patient based on the HOXC6−DLX1-TDRD1 score. AHOXC6−DLX1-TDRD1 score may be generated by determining the area underthe curve (AUC) of a Receiver Operating Characteristic (ROC) curve andcorresponding 95% confidence intervals (CI).

As used herein, the term “individual,” which may be used interchangeablywith the terms “patient” or “subject,” refers to one who receivesmedical care, attention or treatment and may encompass a human patient.As used herein, the term “individual” is meant to encompass a person whohas a prostate cancer, is suspected of having prostate cancer, or is atrisk for developing a prostate cancer. Suitable individuals may include,but are not limited to, individuals having a serum prostate-specificantigen (sPSA) level of at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 ng/ml, or individuals having a sPSA levelwithin a range bounded by any of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 ng/ml (e.g., individuals having a sPSA levelwithin a range of 4-10 ng/ml). The presently disclosed methods, devices,combinations, kits, and systems may relate to detecting elevated sPSA ina sample from an individual.

The term “sample” should be interpreted to include, but not be limitedto, bodily fluids (e.g., blood products including serum) and urine, aswell as tissue samples (e.g., a prostate biopsy). The term sample shouldbe interpreted to include, but not be limited to, serum samples having aserum prostate-specific antigen (sPSA) level of at least about 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng/ml, or serumsamples having a sPSA level within a range bounded by any of 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng/ml (e.g., serumsamples having a sPSA level within a range of 4-10 ng/ml).

The disclosed methods, devices, combinations, kits, and systems mayinclude or utilize means and/or components for detecting mRNA,including, but not limited to mRNA of the genes DLX1, HOXC6, TDRD1,and/or KLK3. Means and components may include, but are not limited toone or more oligonucleotides that hybridize to mRNA, including, but notlimited to mRNA of the genes DLX1, HOXC6, TDRD1, and/or KLK3 or thathybridize to reverse transcribed mRNA (e.g., cDNA), including but notlimited to cDNA of the genes DLX1, HOXC6, TDRD1, and/or KLK3.Oligonucleotides may include a DNA primer form reverse transcribingmRNA, including, but not limited to mRNA of the genes DLX1, HOXC6, TDRD1and/or KLK3. Oligonucleotides may include a pair of DNA primers foramplifying reverse transcribed mRNA (e.g., cDNA), including but notlimited to cDNA of the genes DLX1, HOXC6, TDRD1 and/or KLK3 Means andcomponents may include enzymes for reverse transcribing mRNA (e.g., areverse transcriptase which optionally may be stable at temperatures>70°C.) and/or enzymes for amplifying reverse transcribed mRNA (i.e., DNApolymerases which optionally may be stable at temperature>70° C.).

The disclosed methods, devices, combinations, kits, and systems mayinclude or utilize therapies or therapeutic agents for treating prostatecancer. Therapies for prostate cancer may include, but are not limitedto performing surgery (e.g., surgery to remove cancerous prostatetissue), administering radiation therapy (e.g., radiation therapydirected at cancerous prostate tissue), administeringradiopharmaceutical therapy (e.g., administering radiopharmaceuticalssuch as radio-labelled antibodies directed against cancerous prostatetissue), administering hormone therapy (e.g. administering anti-androgentherapy), administering chemotherapy, administering biologic therapy,administering bisphosphonate therapy, administering cryotherapy (e.g.,cryotherapy directed against the cancerous prostate tissue),administering high-intensity focused ultrasound therapy (e.g.,high-intensity focused ultrasound therapy directed against the cancerousprostate tissue), administering proton beam radiation therapy (e.g.,proton beam radiation therapy directed against the cancerous prostatetissue), or a combination thereof.

The disclosed methods, devices, combinations, kits, and systems and mayincorporate methods, devices, combinations, kits, and systems a known inthe art. (See, e.g., WO 2010/037735; WO 2012/152811; WO2013/064636; andWO 2015/022164; the contents of which are incorporated herein byreference in their entireties.

Example

This example shows the validation of a gene panel based mRNA testperformed on whole urine, and a predictive model (using biomarkersHOXC6, DLX1, TDRD1, and HOXC4) useful for identifying patients withhigh-grade PCa (Gleason score≧7) upon prostate biopsy.

Material and Methods

Study Population In two prospective multicenter studies, men who werescheduled for (initial or repeat) prostate biopsies, based on anelevated sPSA levels (≧3 ng/ml), an abnormal DRE and/or a family historyof PCa were consecutively included. Urine samples were collected after astandardized DRE. Subjects were enrolled from six urology clinics in theNetherlands between September 2009 and July 2011 (Clinical trial A) andbetween July 2011 and September 2014 (Clinical trial B). Exclusioncriteria were: history of PCa, medical therapy known to affect sPSAlevels, prostate biopsy within three months prior to enrolment, andinvasive treatment for benign prostate hyperplasia (BPH) within sixmonths prior to enrolment. Transrectal ultrasound (TRUS) guided prostatebiopsy (median of 10 cores) were performed and evaluated per hospital'sstandard procedure and by local pathologists. Institutional ReviewBoards of all hospitals approved the study protocols and writteninformed consent was obtained from each participant. Test results werenot provided to the clinical sites for patient care and the laboratorytechnicians that performed the biomarker tests were blinded for patientcharacteristics.

Sample Collection and Processing

Approximately 30 ml of first voided urine was collected in a collectioncup after DRE. After collection, the urine was immediately transferredinto a urine specimen transport tube (Gen-Probe, Hologic). The sampleswere shipped at room temperature to a central laboratory, and werestored within 7 days at −80° C.

Laboratory Developed Test (LDT) Development

In the discovery and initial validation study urinary sediments wereused; we subsequently developed and standardized the assay using fixedwhole urine as substrate. The assays were performed using the prototypeamplification kit (Labo Bio-medical Products BV, LBP). In short, RNA wasisolated out of 1 ml of urine using the MagNAPure 96 instrument (Roche).Subsequently, the RNA levels of HOXC4, HOXC6, TDRD1, DLX1, KLK3 and PCA3were determined using one-step reverse transcription quantitative PCR(RT-qPCR).

Next to the expression levels (copy numbers) of the single biomarkers,also the ratio of the biomarkers to KLK3 was determined using the DeltaDeltaCT method (ΔΔCT).

RNA Extraction

Automated nucleic acid isolation was carried out using the MagNA Pure 96Instrument (Roche). In duplicate, RNA was extracted from 1 ml ofprocessed urine specimens with the DNA and Viral NA Large volume kit(Roche) using the Pathogen Universal protocol according to themanufacturer's instructions. Purified RNA (50 μl) from each duplicateextraction was transferred to one well in an 8-well strip (Ambion) andmixed (100 μL total volume). The 8-well strips were stored at −80° C.prior to RT-qPCR determination.

One Step Reverse Transcription Quantitative PCR (RT-qPCR)

A 4-gene RT-qPCR test for use with post-DRE whole urine specimen wasused. The test is based on Taqman® PCR technology combined with theabsolute quantification of target RNA sequences of KLK3, HOXC6, DLX1,TDRD1 and HOXC4. The components for the test include a singleplexmastermix for the 5 targets, a calibrator and a RT-control. Thecalibrator consists of a multi-target-plasmid containing the targetsequences and the RT-control is a mix of in vitro transcribed (IVT) RNAfrom the targeted sequences. Per test run, 10,000 copies of themulti-target-plasmid (Cal 10⁴) and IVT RNA were used as positivereference controls. A separate RT-qPCR assay was developed to measurePCA3 transcripts in urine based on the same principle as describedabove. [assay characteristics not shown]

Quantification of the RNA

For each transcript, 14 μl of RNA specimen or control was mixed with 6μl Mastermix in a single well of a LightCycler® 480 multiwell plate(Roche). The 20 μl reaction was reverse-transcribed and amplified in aLightCycler® 480 II system (Roche) according to the manufacturer'sinstructions of the prototype amplification kit. The levels of geneexpression were calculated with the Abs Quant/2nd Derivative (highconfidence) crossing point (Cp) method implemented by the LightCycler®480 software version 1.5.0 (Roche). The Cross point cycle (Cp; alsoknown as Threshold cycle C_(t)) is the cycle number at which thefluorescence signal crosses the threshold of the baseline in the qPCR.The Cp value inversely correlates with the initial concentration of themRNA, e.g. the lower the Cp the higher the gene expression in thesample. The Cp values and copy numbers of the single target genes weredetermined and the ratio of the target gene versus the reference gene(KLK3) was calculated to normalize the qPCR results, using the DeltaDeltaCp method (ΔΔCp):

Ratio = 2((AvgCp_((Cal 10⁴)_(target)) − Cp  _((sample)_(target))) − (AvgCp_((Cal 10⁴)) − Cp_((sample)_(reference)))) × 10.000

Evaluation Assay Performance

Assay performance was evaluated during the in-study validation. In eachrun a negative nucleic acid isolation control (NAI−) and a negativetemplate control (NTC) were included. The Cp values for the negativecontrols were required to be negative for each of the RT-qPCR assays.KLK3 was used as an endogenous positive nucleic acid isolation controland was required to be positive in each urine sample. In each RT-qPCRrun the positive reference controls were included and monitored. Theywere required to be within a 30% CV compared to the reference value. Theassays for HOXC6, DLX1, TDRD1, HOXC4, KLK3 and PCA3 met these acceptancecriteria parameters in both clinical trials.

Statistical Analysis

Statistical analysis was performed with SPSS® version 20.0. Two-sidedp-values of <0.05 were considered to indicate statistical significance.Nonparametric statistical tests were used and the distribution was givenin median and range (first quartile (Q1)-third quartile (Q3)). Theexpression levels of the biomarkers were compared with the outcome ofthe prostate biopsies. Backward logistic regression analysis was used totest if the novel biomarkers had independent additional predictivevalue. The test performance characteristics, area under the curve (AUC)of the Receiver Operating Characteristic (ROC) curve and corresponding95% confidence intervals (CI) of the final model were determined.Bootstrapping analysis was performed for internal validation of themodels and to test the robustness of the models in predicting high-gradePCa upon biopsy.

Results Patient Characteristics

In total 905 urine samples were collected in two independent prospectiveclinical trials (cohort A: n=519; cohort B: n=386). Patientcharacteristics are shown in Table 1. In cohort A 212 of 519 men (41%)had a positive biopsy outcome of which 109 men (51%) had high-grade PCa(GS≧7). In cohort B this was 181 of 386 men (47%), and 91 (50%) withhigh-grade PC, respectively. There was a statistically significantdifference in the number of patients having previous biopsies (<0.01),with a higher percentage in cohort A as compared to cohort B. No otherstatistically significant differences in baseline characteristics werefound between the cohorts.

TABLE 1 Patient characteristics Cohort A Cohort B (n = 519) median (n =386) median Descriptives (range)/n (%) (range)/n (%) p-value Age  65(44-86)  65 (39-84) 0.28^(a) sPSA (ng/ml)   7.4 (5.5-11.1)   7.3(5.2-10.9) 0.29^(a) PCa in family 91 (18%) 74 (19%) 0.53^(b) No previousbiopsies 410 (79%)  342 (89%)  <0.01^(b) TRUS prostate  48 (15-200)  45(15-270) 0.08^(a) volume (cc) PCa upon biopsy* 214 (41%)  181 (47%) 0.09^(b) GS ≦6 103 (49%)  90 (50%) GS 7 58 (27%) 52 (29%) GS 8-10 51(24%) 39 (22%) DRE = Digital Rectal Examination, TRUS = TransRectalUltraSound, GS = Gleason score *from 2 subjects the total GS could notbe determined, at least a Gleason 4 component was present.^(a)Mann-Whitney U test, significance level <0.05; ^(b)Chi-square test,significance level <0.05

Biomarker Characteristics

All 905 urine samples contained detectable levels of theprostate-specific transcript KLK3 The median biomarker levels (and Cpvalues) were within comparable ranges, with the note that a lower Cpvalue means higher transcript/RNA (Table 2).

TABLE 2 Biomarker characteristics Cohort A Cohort B (n = 519) (n = 386)Copy numbers Cp value Copy numbers Cp value KLK3  19149 (6693-50691)25.3 (23.8-26.8)  20627 (9073-45958) 25.0 (23.8-26.2) PCA3 2140(572-8062) 28.1 (26.1-30.0) 2247 (633-8206) 28.0 (26.1-30.0) HOXC6 96(36-229) 34.2 (32.8-35.7) 100 (37-247)  34.1 (32.7-35.6) DLX1 15 (5-44) 34.9 (32.7-36.2) 13 (4-29)  35.7 (34.6-36.7) TDRD1 73 (29-189) 32.0(30.4-33.3) 64 (25-135) 32.8 (31.7-34.0) HOXC4 76 (34-186) 33.0(31.7-34.2) 79 (31-202) 32.9 (31.5-34.2) median (Q1-Q3) Cp = crossingpoint, Q1-Q3 = inter quartile range

Performance of the Expression of the Single Target Genes Compared to theTarget Gene/KLK3 Ratio

To determine the value of normalization of the expression of the targetgenes with KLK3 expression for diagnosis of high-grade PCa in wholeurine, the AUCs were compared in cohort A. Using the ratio of the targetgenes and KLK3 resulted in an enormous increase of the AUC as is shownfor HOXC6 and DLX1 in FIGS. 1 and 2. For HOXC6 the AUC increased from0.54 to 0.72 for the HOXC6/KLK3 ratio [95% CI: 0.67-0.78]. For DLX1alone the AUC was 0.51 and for the DLX1/KLK3 ratio 0.64 [95% CI:0.58-0.71].

Selection of a Predictive Model for the Diagnosis of High-Grade PCa UponBiopsy

To develop a predictive model the 519 samples derived from cohort A wereused. To compare the utility of the biomarkers to predict high-grade(GS≧7) PCa upon biopsy, a threshold of ≈90% sensitivity was chosen. Thecut-off at ≈90% sensitivity was determined, as were the AUC,sensitivity, specificity, NPV, and PPV for each biomarker (Table 3). Ofthe single markers, HOXC6 had the highest AUC (AUC=0.73 [95% CI:0.68-0.79]) and the highest specificity of 33% at 91% sensitivity.

Biomarker levels of HOXC6 and HOXC4 were observed to strongly correlatewith each other (Pearson correlation (R2) of 0.80) indicating that theywould not complement each other. To determine whether DLX1 and TDRD1could complement the performance of either HOXC6 or HOXC4, models werebased on the sum of the ratios. Using a cut-off of 27.5, the combinationHOXC6+DLX1 had the best performance with an AUC of 0.76 (95% CI:0.71-0.81). The addition of other markers to this model did not resultin an improvement of the test performance (Table 3).

TABLE 3 Biomarker models used for the development with cut-off andclinical performance. Model Cut-off AUC Se (%) Sp (%) NPV (%) PPV (%)PCA3 35.0 0.65 91 20 89 23 TDRD1 1.0 0.69 90 11 80 21 DLX1 0.5 0.65 8316 79 21 HOXC4 15.5 0.64 91 22 90 23 HOXC4 + DLX1 26.5 0.70 91 31 93 25HOXC4 + TDRD1 50.5 0.72 91 30 93 25 HOXC4 + DLX1 + TDRD1 57.5 0.73 91 3193 26 HOXC6 19.5 0.73 91 33 93 26 HOXC6 + DLX1 27.5 0.76 91 36 94 27HOXC6 + TDRD1 50.5 0.74 91 35 94 27 HOXC6 + DLX1 + TDRD1 55.5 0.74 91 3493 26 HOXC6 + HOXC4 + 85.5 0.74 91 33 93 26 DLX1 + TDRD1 AUC = AreaUnder the Curve. Se = Sensitivity. Sp = Specificity. NPV = NegativePredictive Value. PPV = Positive Predictive Value.The combination of HOXC6+DLX1 ratios had the highest AUC overall forprediction of high-grade PCa upon biopsy, this model will be referred toas the HOXC6−DLX1 score.

Bootstrap Analysis

Bootstrap resampling was used as an internal validation of the models.After 100 bootstrap replications, HOXC6 and DLX1 significantly improvedthe predictive accuracy for high-grade PCa in 97% and 98% of thebootstrap samples, respectively. In the biomarker models based on HOXC6,TDRD1 improved the accuracy in 38% of the bootstrap samples. Overall,the models based on HOXC6 had the highest AUCs, and the highest AUCoverall was obtained for the model based on the sum of HOXC6+DLX1.Therefore, the combination of HOXC6+DLX1 was the best predictive modelfor identification of high-grade PCa upon biopsy, this model will bereferred to as the HOXC6−DLX1 score.

Informative Rate

KLK3 was used as a measure for the presence of prostate derivedtranscripts and the expression level was about 1000 fold higher than ofHOXC6 and DLX1 (Table 2). To exclude false negative outcomes, a urinesample with HOXC6−DLX1 score≦27.5 needed to have at least 10,000 copiesof KLK3. As this KLK3 threshold was set, 27 samples in cohort A weremarked as non-informative, resulting in 492 informative samples (95%).In cohort B, 15 samples were marked as non-informative, resulting in 371informative samples (96%).

Clinical Performance of the HOXC6−DLX1 Score for Prediction ofHigh-Grade PCa

To demonstrate the predictive accuracy in cohort A (n=492) ROC curveswere generated for the HOXC6−DLX1 score and PCA3 using high-grade PCaupon biopsy as clinical outcome (FIG. 3). The HOXC6−DLX1 score (AUC 0.75[95% CI: 0.70-0.80]) outperformed PCA3 (AUC 0.65 [95% CI: 0.59-0.71]).The specificity of the HOXC6−DLX1 score was higher compared to PCA3, 32%versus 19%.

Using the 371 informative samples of cohort B, the predictive accuracyof the HOXC6−DLX1 score was independently validated. The clinicalperformance of the HOXC6−DLX1 score in cohort B (AUC 0.73 [95% CI:0.67-0.78]) was comparable to its performance in cohort A (AUC 0.75).Moreover, the sensitivity remained 92% and the specificity increased to37%, versus 32% in cohort A (Table 4).

TABLE 4 Clinical performance of the HOXC6-DLX1 score Cohort A Cohort BHOXC6-DLX1 score (n = 492) (n = 371) AUC 0.75 0.73 (95% CI) (0.70-0.80)(0.67-0.78) Sensitivity 92% 92% Specificity 32% 37% NPV 93% 94% PPV 27%32% AUC = Area Under the Curve, NPV = Negative Predictive Value, PPV =Positive Predictive Value.

HOXC6−DLX1 Scores in Relation to Biopsy Gleason Scores

In cohort A 286 men (58%) had no PCa upon prostate biopsy, 98 men (20%)had GS≦6, and 108 men (22%) had GS≧7. In cohort B this was 196 (53%), 86(23%), and 89 (24%), respectively. The HOXC6−DLX1 score wassignificantly correlated with the Gleason score upon biopsy in bothcohorts A and B (FIG. 4).

Performance of the HOXC6−DLX1 Score in the sPSA ‘Grey Zone’

To determine the additional value in the sPSA ‘grey zone’ (sPSA 4.0-10.0ng/ml), the AUCs of the HOXC6−DLX1 score for diagnosis of high-grade PCawere compared to the AUCs of sPSA in low sPSA subgroups (FIG. 5). TheAUCs of the HOXC6−DLX1 score ranged from 0.68 to 0.71 in cohort A. TheAUCs of sPSA at lower sPSA cut-offs decreased to 0.52. In cohort B theAUCs of the HOXC6−DLX1 score ranged from 0.69 to 0.71, whereas the AUCof sPSA decreased to 0.55 in the lowest sPSA group.

DISCUSSION

In many studies new promising PCa-specific biomarkers have beenidentified, however, to date only few biomarkers have reached clinicalpractice. The challenge is to independently validate the performance ofthe biomarkers in a clinical cohort. In the present example, amulticenter study a model is outlined using promising biomarkers. TheHOXC6−DLX1 score had the best predictive value for high-grade PCa uponprostate biopsy and was validated in an independent cohort.

To prevent false negative samples in this study, a threshold for KLK3level (<10,000 copies) was established for samples with a HOXC6−DLX1score≦27.5. Another explanation for a false negative HOXC6−DLX1 testcould be too little tumor content released into the urine, due to smalltumor size or tumor location (base of the prostate, which cannot bereached by DRE). Urine samples were collected using the Gen-Probe(Hologic) urine tubes.

Currently, no test is completely accurate in detecting PCa. Defining anoptimal cut-off score will always be a compromise between sensitivityand specificity, depending on what risk of missing significant tumors isclinically acceptable. In this example, a sensitivity of 90% forprediction of high-grade PCa upon prostate biopsy was chosen for modeldevelopment.

In cohort A 131 (27%) subjects had a HOXC6−DLX1 score of ≦27.5 and incohort B 111 (30%) of men. Of these men 9 (7%), respectively 7 (6%) hadhigh-grade PCa upon biopsy. In total (n=863), 242 biopsies (28%) couldhave been avoided if the HOXC6−DLX1 score would have been performed,with the risk of missing 7% high-grade PCa.

Nowadays, using sPSA with threshold 4 ng/ml in PCa detection also acertain percentage of the high-grade PCa is missed. In the ProstateCancer Prevention Trial, diagnosis of PCa is reported in 15.2% of menwith a sPSA level of ≦4 ng/ml, of which 14.9% had a high-grade PCa(GS≧7). This risk was very low for patients with a sPSA level<1 ng/ml,but increased to 9.4% in patients with a sPSA between 3-4 ng/ml, i.e.one could conclude that the current accepted risk of missing significantcancers using sPSA is up to 9.4% when a threshold of 4 ng/ml is used, or5.7% when a threshold of 3 ng/ml is used. Using a cut-off of 27.5 forthe HOXC6−DLX1 score, 4% of patients with high-grade PCa in the sPSArange between 4-10 mg/ml would be missed, which is lower than the riskprofile for a sPSA threshold between 2-4 ng/ml (7.2%). Given thecharacteristics of the HOXC6−DLX1 score, the risk to miss high-gradecancer in patients with sPSA values<4 ng/ml might be further reduced.Patients with sPSA levels≦4 ng/ml would be an interesting population tostudy the additional value of the HOXC6−DLX1 urine test.

CONCLUSION

The HOXC6−DLX1 urine test is a powerful tool for prediction ofhigh-grade PCa upon prostate biopsy and could therefore be used indecision making, reducing the number of unnecessary prostate biopsiesand potential overtreatment.

REFERENCES

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In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references may be madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1. A method comprising: (a) reacting a nucleic acid isolated from urineof a patient with a reagent composition that comprises polynucleotidereagents for detecting levels of DLX1 mRNA, HOXC6 mRNA, and KLK3 mRNA inthe nucleic acid sample; and (b) detecting the levels of DLX1 mRNA,HOXC6 mRNA, and KLK3 mRNA in the nucleic acid sample.
 2. The methodaccording to claim 1, wherein the reagent composition further comprisespolynucleotide reagents for detecting levels of TDRD1 mRNA in thenucleic acid sample, and the method further comprises detecting thelevels of TDRD1 mRNA in the nucleic acid sample.
 3. The method accordingto claim 1, further comprising performing a biopsy of prostate tissuefrom the patient.
 4. The method according to claim 1, further comprisingadministering treatment for aggressive prostate cancer.
 5. The methodaccording to claim 4, wherein the treatment for prostate cancercomprises performing surgery, administering radiation therapy,administering radiopharmaceutical therapy, administering hormonetherapy, administering chemotherapy, administering biologic therapy,administering bisphosphonate therapy, administering cryotherapy,administering high-intensity focused ultrasound therapy, administeringproton beam radiation therapy, or a combination thereof.
 6. The methodaccording to claim 1, further comprising: (a) reacting a serum samplefrom the patient with a reagent composition that comprises componentsfor detecting levels of prostate-specific antigen (sPSA) in the serumsample; (b) detecting the levels of sPSA in the serum sample.
 7. Themethod according to claim 1, wherein the polynucleotide reagents fordetecting levels of DLX1 mRNA, HOXC6 mRNA, and KLK3 mRNA in the nucleicacid sample comprise oligonucleotides that hybridize to DLX1 mRNA, HOXC6mRNA, and KLK3 mRNA.
 8. The method accordingly to claim 1, wherein thepolynucleotide reagents for detecting levels of DLX1 mRNA, HOXC6 mRNA,and KLK3 mRNA in the nucleic acid sample comprise a pair ofoligonucleotides for amplifying reverse transcribed mRNA of the genesDLX1, HOXC6 and KLK3.
 9. The method accordingly to claim 1, wherein theurine sample is a first voided urine sample collected after a digitalrectal examination.
 10. The method accordingly to claim 1, furthercomprising isolating the nucleic acid sample from urine of the patient.11. A method comprising detecting mRNA in a urine sample from a patient,wherein the detected mRNA comprises: (a) DLX1 mRNA; (b) HOXC6 mRNA; and(c) KLK3 mRNA.
 12. The method according to claim 10, wherein thedetected mRNA further comprises TDRD1 mRNA.
 13. The method accordinglyto claim 11, wherein: (a) DLX1 mRNA in the urine sample is detected byconverting DLX1 mRNA in the urine sample to cDNA and detecting the DLX1cDNA; (b) HOXC6 mRNA in the urine sample is detected by converting HOXC6mRNA in the urine sample to cDNA and detecting the HOXC6 cDNA. (c) KLK3mRNA in the urine sample is detected by converting KLK3 mRNA in theurine sample to cDNA and detecting the KLK3 cDNA.
 14. The methodaccording to claim 13, wherein the cDNA is detected in step (a), step(b), and step (c) by amplifying the cDNA by performing a polymerasechain reaction.
 15. The method according to claim 11, wherein the urinesample is a first voided urine sample collected after a digital rectalexamination.
 16. The method accordingly to claim 11, further comprisingisolating the nucleic acid sample from urine of the patient.
 17. Amethod comprising: (a) isolating RNA from a urine sample; (b) convertingthe isolated RNA to cDNA; (c) contacting the cDNA with an array; (d)amplifying the cDNA; and (i) detecting DLX1 cDNA; (ii) detecting HOXC6cDNA; and (iii) detecting KLK3 cDNA.
 18. The method according to claim17, further comprising detecting TDRD1 cDNA.
 19. A device, combination,kit, or system comprising: (a) components for obtaining RNA from a urinesample of a patient; and (b) components for detecting mRNA of the genesDLX1, HOXC6 and KLK3 in the obtained RNA.
 20. The device, combination,kit, or system of claim 19, wherein the components for detecting mRNA ofthe genes DLX1, HOXC6 and KLK3 comprise oligonucleotides that hybridizeto mRNA of the genes DLX1, HOXC6 and KLK3.
 21. The device, combination,kit, or system of claim 19, wherein the components for detecting mRNA ofthe genes DLX1, HOXC6 and KLK3 comprise pairs of oligonucleotides foramplifying reverse transcribed mRNA of the genes DLX1, HOXC6 and KLK3.22. The device, combination, kit, or system of any of claim 19, furthercomprising: (c) components for obtaining the urine sample from thepatient.